HEK293cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 10%FBS (Gibco). Once reaching 70% confluence, the cells were transiently transfected with cDNA constructs using Lipofectamine 2000 (Invitrogen). Cells were harvested 48h after transfection for biochemical analyses. Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS; 50 mM Tris, pH8.0) containing Complete Protease Inhibitor Cocktail (Roche). Samples were left on ice for 30 min and sonicated briefly. The insoluble fraction was removed by centrifugation at 15,000 r.p.m. for 15 min at 4°C. Protein concentration was determined by BCA protein assay kit (Bio-Rad). 2× SDS sample buffer (Bio-Rad) containing5% β-mercaptoethanol (Sigma) was added to equal amounts of protein. Proteins were then separated by 4–15% SDS PAGE (Bio-Rad) and transferred to nitrocellulose membrane (0.45 μm). 5% dried milk in TBST (Tris buffered saline with 0.1% Tween 20) was incubated for blocking, and membranes were applied with specific antibodies as listed in Supplementary Table 1b. After washing with TBST and incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology), the antigen-antibody was detected by chemiluminescence (ECL; Pierce) and X-ray film (GE Healthcare).
